Serum Free and Chemically Defined Platform for the Growth and Propogation of HEK293 Cells and Adenovirus Viral Vector Amplification



Todd Sanderson and Terese Joseph




Viral vector production schemes typically involve propagating seed trains and viral amplification in cultures using serum containing media. Although these serum containing media deliver robust viral vector yields, the serum component has several disadvantages such as variability of performance, high costs, and potential of introducing adventitious agents. HEK293 cultures adapted to serum free conditions provide improvements to scalability, significant cost savings, and reduced regulatory burden. We evaluated serum free, chemically defined (CD) media formulations for adenovirus vector production in the iCELLis® Nano bioreactor.









Growth and Adaptation to Chemically Defined Media 293-H cells were directly adapted to three formulations of CD media, each from a different vendor. Growth was monitored for 12 passages. Doubling times calculated, shown in Figure 1.


Figure 1.


Growth and adaption of 293-H cells in chemically defined media





  • Average doubling time was similar between the control media and media B.
  • Media A was slower than the control media.
  • The average doubling times are between 27 and 34 hours, slow but acceptable.


Optimization of Adenovirus Amplification


Process parameters including MOI and duration of infection (DOI) were optimized in 125 mL shake flasks. Total vector productivity was normalized to the MOI 50, 72 hour control media. This data is shown in Figure 2.


Figure 2.


MOI and DOI optimization to maximize adenovirus productivity





  • Highest productivity for all 3 media formulations was achieved with an MOI of 50 and 72 hour DOI.
  • Media A resulted in a ~40% increase in productivity over the control media, whereas media B produced only 60% of the control media.


Chemically Defined Media Performance in the iCELLis Nano Bioreactor


Media A and media B were then used to scale production to the iCELLis Nano bioreactor. These media were benchmarked against DMEM contain 10% FBS.


Figure 3.


Comparison of growth rate in the iCELLis Nano bioreactor and control flask





  • The growth rate in the iCELLis Nano bioreactor is slower than the control flask for media B and the DMEM with 10% FBS control media.
  • Metabolic data showed higher glucose consumption in the control flasks compared to iCELLis Nano bioreactor (data not shown).
  • No significant amounts of cells were found in the recirculating media indicating > 95% of the biomass was contained in the packed bed (data not shown).


The bioreactor was infected with Ad5 as described in the methods section. Total productivities were normalized to the productivity in the serum containing control media in the iCELLis Nano bioreactor. Results are shown in Figure 4.


Figure 4.


Ad5 productivity in the iCELLis Nano bioreactor with chemically defined media





  • Overall productivity in media A was significantly higher than the productivity with both media B and the DMEM with 10% FBS control media.
  • Productivity generally consistent in the iCELLis Nano bioreactor and the control flasks for the 2 chemically defined media formulations. Productivity was lower in the iCELLis Nano bioreactor with control media compared to the control flask.
  • The iCELLis Nano bioreactor can be used to produce adenovirus with serum-free, chemically defined media with viral productivities similar to or greater than serum containing media.


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